Mouse Strain-Dependent Difference Toward the Staphylococcus aureus Allergen Serine Protease-Like Protein D Reveals a Novel Regulator of IL-33.

Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.

Acute myeloid leukemia immunopeptidome reveals HLA presentation of mutated nucleophosmin.

Somatic mutations in cancer are a potential source of cancer specific neoantigens. Acute myeloid leukemia (AML) has common recurrent mutations shared between patients in addition to private mutations specific to individuals. We hypothesized that neoantigens derived from recurrent shared mutations would be attractive targets for future immunotherapeutic approaches. Here we sought to study the HLA Class I and II immunopeptidome of thirteen primary AML tumor samples and two AML cell lines (OCI-AML3 and MV4-11) using mass spectrometry to evaluate for endogenous mutation-bearing HLA ligands from common shared AML mutations. We identified two endogenous, mutation-bearing HLA Class I ligands from nucleophosmin (NPM1). The ligands, AVEEVSLRK from two patient samples and C(cys)LAVEEVSL from OCI-AML3, are predicted to bind the common HLA haplotypes, HLA-A*03:01 and HLA-A*02:01 respectively. Since NPM1 is mutated in approximately one-third of patients with AML, the finding of endogenous HLA ligands from mutated NPM1 supports future studies evaluating immunotherapeutic approaches against this shared target, for this subset of patients with AML.

Using Frameshift Peptide Arrays for Cancer Neo-Antigens Screening.

It has been demonstrated that DNA mutations generating neo-antigens are important for an effective immune response to tumors as evident from recent clinical studies of immune checkpoint inhibitors (ICIs). Further, it was shown that frameshift peptides (FSP) generated in tumors from insertions and deletions (INDELs) of microsatellites (MS) in coding region are a very good correlate of positive response to PD1 treatment. However, these types of DNA-sourced FSPs are infrequent in cancer. We hypothesize that tumors may also generate FSPs in transcription errors through INDELs in MS or by exon mis-splicing. Since there are a finite number of predictable sequences of such possible FSPs in the genome, we propose that peptide arrays with all possible FSPs could be used to analyze antibody reactivity to FSPs in patient sera as a FS neo-antigen screen. If this were the case it would facilitate finding common tumor neoantigens for cancer vaccines. Here we test this proposal using an array of 377 predicted FS antigens. The results of screening 9 types of dog cancer sera indicate that cancer samples had significantly higher antibody responses against FSPs than non-cancer samples. Both common reactive FSPs and cancer-type specific immune responses were detected. In addition, the protection of a common reactive FSP was tested in mouse tumor models, comparing to the non-reactive FSPs. The mouse homologs non-reactive FSPs did not offer protection in either the mouse melanoma or breast cancer models while the reactive FSP did in both models. The tumor protection was positively correlated to antibody response to the FSP. These data suggest that FSP arrays could be used for cancer neo-antigen screening.

A cancer vaccine approach for personalized treatment of Lynch Syndrome.

Lynch syndrome (LS) is a cancer predisposition disorder wherein patients have a 70-80% lifetime risk of developing colorectal cancers (CRC). Finding germline mutations in predisposing genes allows for risk assessment of CRC development. Here we report a germline heterozygous frame-shift mutation in the mismatch repair MLH1 gene which was identified in members of two unrelated LS families. Since defects in DNA mismatch repair genes generate frame-shift mutations giving rise to highly immunogenic neoepitopes, we postulated that vaccination with these mutant peptide antigens could offer promising treatment options to LS patients. To this end we performed whole-exome and RNA seq analysis on the blood and tumour samples from an LS-CRC patient, and used our proprietary neoepitope prioritization pipeline OncoPeptVAC to select peptides, and confirm their immunogenicity in an ex vivo CD8+ T cell activation assay. Three neoepitopes derived from the tumour of this patient elicited a potent CD8+ T cell response. Furthermore, analysis of the tumour-associated immune infiltrate revealed CD8+ T cells expressing low levels of activation markers, suggesting mechanisms of immune suppression at play in this relapsed tumour. Taken together, our study paves the way towards development of a cancer vaccine to treat or delay the onset/relapse of LS-CRC.

Towards a vaccine to prevent cancer in Lynch syndrome patients.

Germline mutations of DNA mismatch repair (MMR) genes predispose Lynch syndrome mutation carriers to the development of MMR-deficient tumors. MMR-deficient tumors show high-level microsatellite instability (MSI-H) and are typically characterized by a comparatively favorable prognosis and the absence of distant organ metastasis. Lynch syndrome-associated cancers are characterized by a pronounced local anti-tumoral immune response and usually display dense lymphocyte infiltration. This finding strongly suggested that the immune system may play an active role in the surveillance and biology of these cancers. The progression of MMR deficient cancers seems to be triggered by mutations in microsatellite sequences within gene-encoding regions. These mutations may cause shifts of the translational reading frame and thus give rise to the generation of potentially immunogenic frameshift peptides (FSP) at the carboxy terminal end of the respective gene products. FSP-specific immune responses are thought to represent one major mechanism by which the host's adaptive immune system can recognize and potentially control Lynch syndrome-associated MSI-H cancers. Consequently, vaccination with FSP antigens represent a promising approach for treatment of Lynch syndrome-associated cancers, potentially also suitable for tumor prevention in so far tumor-free Lynch syndrome germ line mutation carriers. This review will summarize the molecular mechanisms contributing to the immunological phenotype of MSI-H cancers. In addition, clinical perspectives will be discussed, focusing on MSI-H cancer-associated FSP antigens as potential targets for immune therapy approaches.

Genome-wide expression profiling identifies an impairment of negative feedback signals in the Crohn's disease-associated NOD2 variant L1007fsinsC.

NOD2 is an intracellular receptor for the bacterial cell wall component muramyl dipeptide (MDP), and variants of NOD2 are associated with chronic inflammatory diseases of barrier organs (e.g., Crohn's disease, asthma, and atopic eczema). It is known that activation of NOD2 induces a variety of inflammatory and antibacterial factors. The exact transcriptomal signatures that define the cellular programs downstream of NOD2 activation and the influence of the Crohn-associated variant L1007fsinsC are yet to be defined. To describe the MDP-induced activation program, we analyzed the transcriptomal reactions of isogenic HEK293 cells expressing NOD2(wt) or NOD2(L1007fsinsC) to stimulation with MDP. Importantly, a clear loss of function could be observed in the cells carrying the Crohn-associated variant L1007fsinsC, whereas the NOD2(wt) cells showed differential regulation of growth factors, chemokines, and several antagonists of NF-κB (e.g., TNFAIP3 [A20] and IER3). This genotype-dependent regulation pattern was confirmed in primary human myelomonocytic cells. The influence of TNFAIP3 and IER3 in the context of NOD2 signaling was characterized, and we could validate the predicted role as inhibitors of NOD2-induced NF-κB activation. We show that IER3 impairs the protective effect of NOD2(wt) against bacterial cytoinvasion. These results further our understanding of NOD2 as a first-line defense molecule and emphasize the importance of simultaneous upregulation of counterregulatory anti-inflammatory factors as an integral part of the NOD2-induced cellular program. Lack of these regulatory events due to the L1007fsinsC variant may pivotally contribute to the induction and perpetuation of chronic inflammation.

Multiple RNA surveillance mechanisms cooperate to reduce the amount of nonfunctional Ig kappa transcripts.

Random V(D)J junctions ensure that the diversity of the Ig primary repertoire is adapted to the vast heterogeneity of Ags. In two-thirds of cases, recombination between variable segments induces a frameshift in the open reading frame and generates a premature termination codon. In B cells harboring biallelic V(D)J rearrangement of Ig genes, transcription is known to occur on both the functional and nonfunctional alleles, generating considerable amounts of primary transcripts with out-of-frame V regions. In this study, we analyzed in cell lines and primary B cells the RNA surveillance of nonfunctional Igkappa transcripts arising from nonproductive rearrangement. We demonstrated that splicing inhibition, nonsense-mediated decay and nonsense-altered splicing each have an individual partial effect that together associate into an efficient surveillance machinery, downregulating nonfunctional Igkappa mRNA. Moreover, we provide evidence that the RNA surveillance efficiency increases throughout B cell development. Whereas splicing inhibition remains constant in most cell lines, differences in nonsense-mediated decay and nonsense-altered splicing are responsible for the higher RNA surveillance observed in plasma cells. Altogether, these data show that nonfunctionally rearranged alleles are subjected to active transcription but that multiple RNA surveillance mechanisms eradicate up to 90% of out-of-frame Igkappa mRNA.

Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences.

CD8+ cytotoxic T lymphocyte (CTL)-mediated immune responses to HIV contribute to viral control in vivo. Epitopes encoded by alternative reading frame (ARF) peptides may be targeted by CTLs as well, but their frequency and in vivo relevance are unknown. Using host genetic (human leukocyte antigen [HLA]) and plasma viral sequence information from 765 HIV-infected subjects, we identified 64 statistically significant (q<0.2) associations between specific HLA alleles and sequence polymorphisms in alternate reading frames of gag, pol, and nef that did not affect the regular frame protein sequence. Peptides spanning the top 20 HLA-associated imprints were used to test for ex vivo immune responses in 85 HIV-infected subjects and showed responses to 10 of these ARF peptides. The most frequent response recognized an HLA-A*03-restricted +2 frame-encoded epitope containing a unique A*03-associated polymorphism at position 6. Epitope-specific CTLs efficiently inhibited viral replication in vitro when viruses containing the wild-type sequence but not the observed polymorphism were tested. Mutating alternative internal start codons abrogated the CTL-mediated inhibition of viral replication. These data indicate that responses to ARF-encoded HIV epitopes are induced during natural infection, can contribute to viral control in vivo, and drive viral evolution on a population level.

NKp44 expression, phylogenesis and function in non-human primate NK cells.

Molecular and functional characterization of the natural cytotoxicity receptor (NCR) NKp44 in species other than Homo sapiens has been elusive, so far. Here, we provide complete phenotypic, molecular and functional characterization for NKp44 triggering receptor on Pan troglodytes NK cells, the closest human relative, and the analysis of NKp44-genomic locus and transcription in Macaca fascicularis. Similar to H. sapiens, NKp44 expression is detectable on chimpanzee NK cells only upon activation. However, basal NKp44 transcription is 5-fold higher in chimpanzees with lower differential increases upon cell activation compared with humans. Upon activation, an overall 12-fold lower NKp44 gene expression is observed in P. troglodytes compared with H. sapiens NK cells with only a slight reduction in NKp44 surface expression. Functional analysis of 'in vitro' activated purified NK cells confirms the NKp44 triggering potential compared with other major NCRs. These findings suggest the presence of a post-transcriptional regulation that evolved differently in H. sapiens. Analysis of cynomolgus NKp44-genomic sequence and transcription pattern showed very low levels of transcription with occurrence of out-of-frame transcripts and no surface expression. The present comparative analysis suggests that NKp44-genomic organization appears during macaque speciation, with considerable evolution of its transcriptional and post-transcriptional tuning. Thus, NKp44 may represent an NCR being only recently emerged during speciation, acquiring functional relevance only in non-human primates closest to H. sapiens.

Out of frame peptides from BCR/ABL alternative splicing are immunogenic in HLA A2.1 transgenic mice.

New, potentially tumor-specific antigens have been described in Bcr/Abl positive leukemias. Besides the main BCR/ABL hybrid fusion transcripts, a small number of transcripts derived from alternative splicing between BCR exons 1, 13, and 14 with ABL exons 4 and 5 have been identified. These variants are expressed in chronic myelogenous leukemia and acute lymphocytic leukemia patients. The transcriptional products were characterized at their C-terminus by a large amino acid portion derived from out of frame (OOF) reading of the ABL gene. This OOF peptide is expressed only in leukemic cells and has no homology with known human proteins. In order to study an in vivo model, three 39-amino acid peptides, each corresponding to a third of the whole human OOF peptide sequence, were tested for their capacity to elicit specific immune responses in HLA A2.1 transgenic mice. Peptides A and B, but not C, induced the production of specific antisera, while A and C induced the generation of specific cytotoxic T lymphocytes.

Prediction of the immunogenic potential of frameshift-mutated antigens in microsatellite instable cancer.

Microsatellite instable (MSI) cancers express frameshift-mutated antigens, the C-terminal polypeptides of which are foreign to the immune system. Consequently, these antigens constitute a unique pool of tumor-specific antigens that can be exploited for patient diagnosis and selective, immune-mediated targeting of cancers. However, other than their sequence, very little is known about the characteristics of the majority of these proteins. We therefore developed a methodology for predicting their immunogenic behavior that is based on a gene-expression system, in which each of the proteins was fused to a short C-terminal polypeptide comprising two epitopes that can be readily detected by T-cells and antibodies, respectively. In this manner, accumulation of the antigens and processing of peptides derived thereof into MHC can be monitored systematically. The antigens, which accumulate in the cells in which they are synthesized, are of primary interest for cancer immunotherapy, because peptide epitopes derived thereof can be presented by dendritic cells in addition to the tumor cells themselves. As a result, these antigens constitute the best targets for a coordinated immune response by both CD8+ and CD4+ T-cells, which increases the likelihood that tumor-induced immunity would be detectable against these antigens in cancer patients, as well as the potential value of these antigens as components of anticancer vaccines. Our data indicate that, of 15 frameshift-mutated antigens examined in our study, 4 (TGFbetaR2-1, MARCKS-1, MARCKS-2 and CDX2-2) are of primary interest, and 4 additional antigens (TAF1B-1, PCNXL2-2, TCF7L2-2 and Baxalpha+1) are of moderate interest for further tumor immunological research.

Identification of two pseudogenes with sequence homology to human and gorilla MHC class IA genes: ancestral haplotype in the Filipino population.

While characterizing exons 2 and 3 of the class I human leukocyte antigen (HLA)-A locus in human lymphocytes, two similar but unexpected PCR products were detected in six samples of Filipino ethnicity. A nucleotide sequence analysis of the two amplicons, tentatively named HLA-COQ and HLA-DEL, rendered them as two novel and seemingly related sequences, both with homology to the gorilla and human major histocompatibility complex (MHC) A locus. Exon 2 is similar to the published human pseudogenes HLA-BEL, HLA-Y, and to primate MHC Gogo-A*0501, differing by 2 bp from HLA-BEL, and HLA-Y, and by 4 bp from Gogo-A*0501. Exon 3 is most similar to HLA-A*2902 and A*310102, differing by 7 bp from A*2902, and by 8 bp from A*31012. Genomic sequence comparison of exons 1 to 8 indicates that their closest published match is to the Gogo-A*0501. Complete typing at the HLA-A, -B, -C, DRB1, and DRB5 loci for the six samples yielded the reoccurring types: HLA-A*3401, -B*1521/1525, -Cw*0403, -DRB1*150201, and DRB5*010101. Thus far, HLA-COQ and HLA-DEL have been detected only in Filipino samples containing these HLA types. The HLA-COQ gene is nonfunctional based on a stop codon located in exon 4. HLA-DEL is also a nonfunctional gene because of the dual cytosine insertion in exon 4, with a reading frame shift generating a stop codon downstream. Parsimony analysis of the two pseudogenes with 31 other primate A locus coding regions resulted in a phylogenetic tree that segregated the two pseudogenes with the Gogo-A*0501, suggesting that HLA-COQ, HLA-DEL, and Gogo-A*0501 evolved from a common ancestral allele.

T cells associated with tumor regression recognize frameshifted products of the CDKN2A tumor suppressor gene locus and a mutated HLA class I gene product.

The dramatic tumor regression observed following adoptive T cell transfer in some patients has led to attempts to identify novel Ags to understand the nature of these responses. Nearly complete regression of multiple metastatic melanoma lesions was observed in patient 1913 following adoptive transfer of autologous tumor-infiltrating lymphocytes. The autologous 1913 melanoma cell line expressed a mutated HLA-A11 class I gene product that was recognized by the bulk tumor-infiltrating lymphocytes as well as a dominant T cell clone derived from this line. A second dominant T cell clone, T1D1, did not recognize the mutated HLA-A11 product, but recognized an allogeneic melanoma cell line that shared expression of HLA-A11 with the parental tumor cell line. Screening of an autologous melanoma cDNA library with clone T1D1 T cells in a cell line expressing the mutated HLA-A11 gene product resulted in the isolation of a p14ARF transcript containing a 2-bp deletion in exon 2. The T cell epitope recognized by T1D1, which was encoded within the frameshifted region of the deleted p14ARF transcript, was also generated from frameshifted p14ARF or p16INK4a transcripts that were isolated from two additional melanoma cell lines. The results of monitoring studies indicated that T cell clones reactive with the mutated HLA-A11 gene product and the mutated p14ARF product were highly represented in the peripheral blood of patient 1913 1 wk following adoptive transfer, indicating that they may have played a role in the nearly complete tumor regression that was observed following this treatment.

Prevalence of the chemokine receptor CCR5-Delta32 gene mutation in periodontal disease.

A 32-base-pair deletion in the CCR5 gene was previously shown to influence the susceptibility for several infectious diseases. The present study compared the frequency of the CCR5-Delta32 mutation among subjects with periodontal disease and healthy control individuals. The prevalence of the CCR5-Delta32 mutation was determined in 81 patients with generalized periodontitis and 121 healthy controls. Standardized clinical and radiographic criteria were used for the diagnosis of periodontitis for each subject. The CCR5-Delta32 mutation was identified by PCR amplification and subsequent agarose gel electrophoresis. Genotype and allele frequencies among both study groups were compared using Fisher's exact test at a level of significance of 5% (P<0.05). The frequency of the CCR5-Delta32 allele was 9.9% (16/162) for periodontitis patients and 10.7% (26/216) for the healthy controls. The allele frequencies between periodontitis patients and the control group for the CCR5-Delta32 mutation were not significantly different (P=0.801). The present study revealed no association between the CCR5-Delta32 mutation and susceptibility to periodontal disease.

Tumor-specific immunological recognition of frameshift-mutated peptides in colon cancer with microsatellite instability.

Colorectal cancers with microsatellite instability (MSI+ CRCs) caused by dysfunction of DNA mismatch repair have unique clinicopathological characteristics including good prognosis with T-cell infiltration in tumor. To identify tumor antigens that induce immune response against MSI+ CRC, SEREX (serological analysis of recombinant cDNA expression cloning) was applied. By screening a lambda phage cDNA library constructed from three MSI+ CRC cell lines with serum from a patient with MSI+ CRC with abundant T-cell infiltrates in tumor, 64 antigens were isolated. Immunogenicity of each antigen was evaluated by screening sera from patients with various cancers and from healthy individuals, and specific IgG antibodies (Abs) for 49 antigens were detected only in MSI+ CRC patients. A frameshift mutation in the repetitive G sequences (microsatellite) in the coding region of CDX2, one of the identified antigens, was found in the tumor tissue of the patient who had anti-CDX2 serum Ab. The Ab recognized both the COOH-terminal tumor-specific peptides created by the frameshift mutation and the NH(2)-terminal normal peptides of CDX2 when Western blot analysis was performed using various bacterial recombinant CDX2 proteins including the normal and altered peptides, which indicated that immune response could be raised against tumor-specific peptides generated through MSI. The anti-CDX2 Ab was detected only in the patient with the CDX2 frameshift mutation in tumor and disappeared 7 years after the curative resection, suggesting that this immune response may also be useful as a tumor marker. No altered subcellular localization and transcription ability was demonstrated in the mutated CDX2, although decreased expression was suggested in immunohistochemical analysis. Therefore, tumor-specific peptides generated by MSI may be involved in antitumor immune responses and may be useful for the development of diagnostic and therapeutic methods for patients with MSI+ CRC.

Non-expression of HLA-B*5111N is caused by an insertion into the cytosine island at exon 4 creating a frameshift stop codon.

The identification of the "blank" allele HLA-B*5111N, which was detected in German and Czech individuals, is described. In the pedigree analysis this new allele segregates with the serological haplotype HLA-A2; B-; DR4 which is frequent in Czech population. The non-expression of B*5111N is caused by the insertion of an additional cytosine molecule at the cytosine island between the nucleotides 621-626 (codons 183-185, first three codons of exon 4) leading to a frame shift that creates a stop codon at codon 196. This insertion may be explained either by conversion with the pseudogene HLA-J or by slipped-strand mispairing. In order not to overlook the presence of alleles with altered expression in case of hematopoietic stem cell transplantation, both serological and DNA-based typing should be performed (Note).

B cell development under the condition of allelic inclusion.

Mice whose IgH alleles are engineered to encode two distinct antibody heavy (H) chains generate a normal-sized B cell compartment in which most cells stably express the two heavy chains. This demonstrates that "toxicity" of bi-allelic H chain expression and cell-autonomous mechanisms of silencing in-frame IgH gene rearrangements do not significantly contribute to allelic exclusion at the IgH locus. Notwithstanding, the stability of the various engineered IgH loci during B cell development in the bone marrow differed substantially from each other.

Recognition of out-of-frame major histocompatibility complex class I-restricted epitopes in vivo.

In the course of constructing a recombinant vaccinia virus encoding the influenza A nucleoprotein (NP) gene preceded by the hemagglutinin leader sequence, we isolated a single base-pair deletion mutant which gave rise to L+NP(1-159) in which only the first 159 amino acids were in frame. Despite this, when we infected target cells, we found that the point mutant was able to sensitize them for lysis not only by cytotoxic T cells recognizing residues 50-58 (the in-frame portion), but also by CTL to epitopes which are downstream of the mutation (366-374 and 378-386). Furthermore, normal C57BL/6 mice can be primed with the frameshift NP to recognize the immunodominant Db-restricted epitope 366-374 (which is out of frame). Experiments in which the mutant gene product was processed in the endoplasmic reticulum of target cells suggested that the apparent suppression occurred during polypeptide extension.